Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.

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